The three common human apolipoprotein E protein isoforms (apoE2, E3 and E4) are encoded by three gene alleles (e2, e3, e4). The e4 allele is a well-established genetic risk factor for the devastating age-related dementia, Alzheimer's disease (AD). Thus, isoform-specific apoE functions correlating it to AD risk remain the subject of intense research effort. One possibility is that the apoE isoforms differentially modulate amyloid-beta peptide (A[unreadable]) function. Currently, there is limited information on the mechanisms underlying structural and functional interactions between apoE and A[unreadable]42. Extensive biochemical evidence shows that apoE interacts with A[unreadable];however, the functional implications at the cellular level remain unclear. We hypothesize that intraneuronal A[unreadable]42 accumulation is exacerbated by apoE4 due its impaired recycling and prolonged intracellular retention compared to the other isoforms, and that these effects involve isoform- specific apoE:A[unreadable] interactions. Therefore, the goal of the proposed research is to determine the role of apoE isoform and interactions between apoE and A[unreadable] in the accumulation of intraneuronal A[unreadable]. We will utilize in vitro primary neuron/glia co-cultures as brain-relevant model systems. The glia are derived from human apoE-targeted replacement mice and express the human apoE isoforms in a native conformation and environment, factors critical for its physiological function. The neurons are derived from "SxFAD" mice (Dr. Robert Vassar), which constitutively produce endogenous A[unreadable]42, or WT neurons in the presence of exogenous oligomeric A[unreadable]42, a soluble aggregate that causes apoE4-potentiated neuron damage. We will first measure intracellular distribution of apoE and A[unreadable], focusing on their subcellular co-localization by density-gradient fractionation and immunofluorescence staining. We will then determine the intra- and extracellular sites of apoE:A[unreadable] interactions, performing initial biochemical characterization using methods to detect both strong and weak associations: SDS-PAGE, coimmunoprecipitation and size-exclusion chromatography. Finally, we will determine the role of endocytic apoE receptors in uptake of apoE and A[unreadable]. Our society is faced with a growing incidence of AD, a neurological disease already placing increasing strain on the health and well-being of our elderly, and on our economy. Critical to overcoming this growing burden is advancing our understanding of the disease onset and progression, and the best pathways to intervene for its prevention and treatment. As proposed in this work, elucidating where and how two key protein players, apoE and A[unreadable], interact in the brain will reveal important mechanistic details essential to understanding their roles in neuron damage, and potentially lead to novel and selective AD therapies.